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Interrogation of the Dynamic Properties of Higher-Order Heterochromatin Using CRISPR/dCas9

ABSTRACT: Interrogation of the Dynamic Properties of Higher-Order Heterochromatin Using CRISPR/dCas9

PROVIDER: PRJNA732641 | ENA |

REPOSITORIES: ENA

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Interrogation of the dynamic properties of higher-order heterochromatin using CRISPR-dCas9.

Project description:Eukaryotic chromosomes feature large regions of compact, repressed heterochromatin hallmarked by Heterochromatin Protein 1 (HP1). HP1 proteins play multi-faceted roles in shaping heterochromatin, and in cells, HP1 tethering to individual gene promoters leads to epigenetic modifications and silencing. However, emergent properties of HP1 at supranucleosomal scales remain difficult to study in cells because of a lack of appropriate tools. Here, we develop CRISPR-engineered chromatin organization (EChO), combining live-cell CRISPR imaging with inducible large-scale recruitment of chromatin proteins to native genomic targets. We demonstrate that human HP1α tiled across kilobase-scale genomic DNA form novel contacts with natural heterochromatin, integrates two distantly targeted regions, and reversibly changes chromatin from a diffuse to compact state. The compact state exhibits delayed disassembly kinetics and represses transcription across over 600 kb. These findings support a polymer model of HP1α-mediated chromatin regulation and highlight the utility of CRISPR-EChO in studying supranucleosomal chromatin organization in living cells.

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Interrogation of the Dynamic Properties of Higher-Order Heterochromatin Using CRISPR/dCas9

Project description:Eukaryotic chromosomes feature large regions of compact, transcriptionally-repressed heterochromatin hallmarked by the presence of Heterochromatin Protein 1 (HP1) family members. HP1 proteins play multi-faceted roles in directly shaping the properties of heterochromatin, and in vivo , HP1 tethering to individual gene promoters leads to epigenetic modifications and gene silencing. However, emergent properties of HP1 at supranucleosomal scales have been difficult to study in cells due to a lack of appropriate tools. Here, we develop CRISPR-Engineered Chromatin Organization (EChO), a novel approach for combining live cell CRISPR-based imaging with inducible and reversible large-scale recruitment of heterochromatin components to native chromatin in human cells. Using CRISPR-EChO, we demonstrate that human HP1α binding across tens of kilobases of genomic DNA leads to formation of novel contacts with other heterochromatin regions and reversibly compacts chromatin from a diffuse to condensed state. The condensed chromatin state exhibits delayed disassembly kinetics and represses transcription across over 600 kilobases. Collectively, these findings support a polymer model of HP1α-mediated chromatin regulation and highlight the utility of CRISPR-EChO in studying and manipulating supranucleosomal chromatin organization in living cells.

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Project description:Abnormal chromatin structure is one of the hallmarks in cancer and has long been used for cancer diagnosis, but the observed aberrant chromatin structures characteristic of cancer cells is limited to microscale features, largely due to due to the diffraction-limited resolution of conventional light microscopy. Recent advances in super-resolution microscopy overcome the technical limitations of conventional light microscopy and enable the in-situ visualization of the higher-order chromatin structure down to ~30 nm. Here we present PathSTORM, a variant of stochastic optical reconstruction microscopy (STORM) optimized for super-resolution imaging of densely packed higher-order chromatin organization at different epigenomic states on pathological tissue. We then apply this tool to characterize the structural alteration of higher-order heterochromatin, enriched with H3K9me3 and satellite repeats, at various stages of carcinogenesis in mouse models and human tissue specimens. PathSTORM reveals that a significant disruption of higher-order heterochromatin structure occurs in early carcinogenesis when tissue still appears histologically normal and becomes progressively severe during neoplastic progression. The disrupted higher-order heterochromatin structure also appears to be a common feature that reflects the evolution of carcinogenesis independent of molecular pathways in multiple tumor types. Taken together, PathSTORM can serve as a powerful tool to visualize higher-order chromatin structure in the spatial context of tissue architecture on pathological tissue. Our findings provide new insights on the role of higher-order heterochromatin structure in carcinogenesis, especially at the early stage, and have the potential for improving cancer diagnosis, risk stratification and facilitating the development and evaluation of new preventive strategies.

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